| Cilium is a subcellular compartment which protrude from cell surface.According to the combination of axial microtubule and cytoskeletal structure(9+2 or 9+0),the cilium can be divided into two different types,which was known as motile cilium and primary cilium(PC)[1].As lack of central microtubule,the PC has no function with cell migration.The PC widely presents in all kind of vertebrate cells and for each cell,there is a single one PC on it [2].It has been reported that primary cilia are involved in regulating several signal transduction pathways,including Hedgehog(Hh),PDGF and WNT pathways [3].Hh signaling pathway is a highly conserved in metazon.It plays important roles in embryogenesis and homeostasis in adult [4].In vertebrates,primary cilia is key for Hh signal transduction.However,how PC delicate control Hh pathway activity is still unclear.Based on our previous studies on GRK2 and other key components of Hh pathway,we hypothesied that PKA activity in PC is probably the key point for regulating Hh pathway.However,because that there is no effective methods and materials for detecting and monitoring small signal molecules in PC,it is impossible for delicate analyzing Hh signaling molecular mechanism and mechanism of other PC associated signaling pathways.To break the bottleneck,we decided to use zebrafish as our model animal to establish stable PC marker and multiple PC involved signal molecules detectable bio-senser.We use the N-terminal of Nephosistin 3(z Nphp3)as PC localization signal peptide and construct z Nphp3N-m Cherry fusion protein to lable PC.We used z Nphp3N-AKAR2 and z Nphp3N-GCa MP6 to detect PKA activity and calcium current involved in PC,respectively.We construct constitutive expressed ubiquitin and β-actin promotor as the promotor of above described fusion gene,that the fusion protein can expressed in all cells.Recently,four of the stable transgenic zebrafish lines are successfully generated.They were Tg(β-actin-z Nphp3N-m Cherry),Tg(β-actin-z Nphp3N-AKAR2),Tg(ubi-z Nphp3N-AKAR2)and Tg(β-actin-z Nphp3N-GCa MP6).These transgenic embryos normally developed,and showed no obvious abnormality phenotype.The transgenic lines are viable.To futher verify the detection function of PKA activity in PC of z Nphp3N-AKAR2,we performed the FRET assay.After treatment of dn PKA,a PKA activity inhibitor,no FRET efficiency was detected.By contrast,high FRET efficiency was detected in embryos treated by foskolin,a PKA agonist.These results suggested that ciliary z Nphp3N-AKAR2 can be used as a PKA activity biosenser.In addition,certain FRET efficiency was detected in the embryos treated with cyclopamine,the antagonist of Hh pathway.This preliminary data on PC activity assay indicated that PC probably regulates Hh signal transduction by modulating its PKA activity.To unveil the Hh pathway mechanism and the pathology of diseases due to aberrant Hh activity.The clinical research was also performed simultaneously.A new Gorlin syndrome family was identified.To confirm this pedigree,the detail clinical examination was performed.Then the whole exome sequencing was perfomed to dissect the potential pathology.A new INDEL mutation on PTCH1 was screened and verified by sanger-sequencing.This new INDEL mutation led early stop codon of PTCH1,one key component of Hh pathway.Further study of this INDEL mutation on PC PKA activity will be performed to provide the delicate pathology of this Gorlin syndrome family.In summary,several transgenic materials for ciliary marker and biosensors were generated and analyzed.And the protocol for ciliary PKA activity assay were established in this work.These materials and methods would strongly help for dissecting the mechanism of PC related signaling pathways and the pathology of PC related human diseases. |
